Palladino et al. Basic and Clinical Andrology (2018) 28:14 https://doi.org/10.1186/s12610-018-0079-x

RESEARCHARTICLE Open Access Effects of lipopolysaccharide-induced inflammation on hypoxia and inflammatory expression pathways of the rat testis Michael A. Palladino*, Genevieve A. Fasano, Dharm Patel, Christine Dugan and Marie London

Abstract Background: Bacterial infection and inflammation of the testis impairs fertility, yet an understanding of inflammatory responses of the testis is incomplete. We are interested in identifying gene pathways involved in the detection and clearance of infectious microbes in the male reproductive tract. In previous studies in our lab focused on hypoxia-responsive of the testis, preliminary experiments suggested that genes classically categorized as hypoxia genes are also activated during antimicrobial responses. The purpose of this study was to identify hypoxia and inflammatory gene pathways that contribute to antimicrobial protection of the testis and to consider possible cross-talk and interactions between these pathways. Inflammation was induced in Sprague-Dawley rats using P. aeruginosa or E. coli lipopolysaccharide (LPS). Levels of hypoxia-inducible factor-1 (HIF-1α) and nuclear factor kappa B (NF-κB) were measured, and hypoxia and inflammatory gene expression patterns in testis were analyzed by gene expression profiling using real-time quantitative PCR arrays. Results: In LPS-treated rats, HIF-1α protein increased with no change in Hif-1α mRNA. Western Blot analysis also demonstrated no change in NF-κB and inhibitory NFKB alpha (IκBα) protein levels following LPS treatment. Five hypoxia pathway genes (Angptl4, Egr1, Ier3, Pai1, and Glut1), and 11 inflammatory pathway genes (Ccl12, Cc13, Cd14, Cxcl10, Icam1, Il10, Il1b, Il6, Nfkbia, Tlr2, Tnf) up-regulated after 3 h of inflammation. Angptl4, Ccl12, Cc13, Cd14, Egr1, Nfkbia, Tlr2, and Tnf remained elevated at 6 h. Six genes were up-regulated at 6 h only (Bhlhe40, C3, Jak2, Nlrp3, Slc11a1, Tlr1). One gene (Tlr5) was down-regulated after 3 h and no genes at 6 h. Electrophoretic mobility shift assay results suggest a decrease in NF-κB binding activity following LPS treatment. Conclusions: Testicular HIF-1α is up-regulated following LPS-induced inflammation. In contrast to other tissues, in which HIF-1α is up-regulated through transcriptional activation via NF-κB, we conclude that HIF-1α in the testis is not up-regulated through an increase in Hif-1α mRNA or through NF-κB-dependent mechanisms. Hypoxia pathway genes and genes involved in Toll-like receptor (TLR) and cytokine-mediated signaling comprise major functional categories of up-regulated genes, demonstrating that both hypoxia and classic inflammatory pathways are involved in inflammatory responses of the testis. Keywords: Hypoxia, Orchitis, Pathogens, Lipopolysaccharide, Inflammation

* Correspondence: [email protected] Note: Co-authors GF, DP, CD and ML were students at Monmouth University when the work was carried out. All have graduated and are currently in graduate school, medical school or in positions in industry Monmouth University, 400 Cedar Avenue, West Long Branch, NJ 07764, USA

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 2 of 12

Résumé Contexte: L’infection et l’inflammation bactériennes du testicule altèrent la fertilité; cependant la compréhension des réponses inflammatoires du testicule est. encore incomplète. Nous nous sommes intéressés à l’identification des voies des gènes impliqués dans la détection et l’élimination des microbes infectieux dans l’appareil reproductif masculin. Dans de précédentes études menées dans notre laboratoire, et centrées sur des gènes sensibles à l’hypoxie, les expérimentations préliminaires suggéraient que les gènes classiquement catégorisés comme gènes de l’hypoxie étaient aussi activés au cours des réponses antimicrobiennes. Le but de la présente étude était d’identifier les voies des gènes qui contribuaient à la protection antimicrobienne du testicule et d’examiner de potentiels intermodulations et interactions entre ces voies. L’inflammation a été induite chez des rats Sprague-Dawley en utilisant des lypopolysaccharides (LPS) de P. aeruginosaet d’E. coli. Les taux de protéine du facteur-1 inductible par l’hypoxie (HIF1- α) et du facteur nucléaire kappa B (NF- kB) ont été mesurés; les profils d’expression des gènes de l’hypoxie et de l’inflammation dans le testicule ont été analysés par profilage de l’expression génique par PCR quantitative en temps réel. Résultats: Chez les rats traités par LPS, la protéine HIF-1 α a augmenté sans modification de Hif-1αmRNA. L’analyse par Western Blot a aussi montré l’absence de modifications des taux de NF-kB et de la protéine inhibitrice NFKB alpha (IkB α) après traitement. Cinq gènes de la voie hypoxie (Angptl4, Egr1, Ier3, Pai1,et Glut1), et 11 gènes de la voie inflammatoire (Ccl12, Cc13, Cd14, Cxcl10, Icam1, Il10, Il1b, Il6, Egr1, Nfkbia, Tlr2,etTnf) ont été régulés à la hausse après 3 heures d’inflammation. Angptl4, Ccl12, Cc13, Cd14, Egr1, Nfkbia, Tlr2,etTnfsont restés élevés à 6 heures. Six gènes n’ont ont été régulés à la hausse qu’à 6 heures (Bhlhe40, C3, Jak2, Nlrp3, Slc11a1, Tlr1). Un gène (Tlr5) a été régulé à la baisse après 3 heures et aucun gène à 6 heures. Les résultats du test de décalage de la mobilité électrophorétique suggèrent une baisse de l’activité de liaison de NF- kB après traitement par LPS. Conclusions: HIF-1α testiculaire est. régulé à la hausse après inflammation induite par LPS. Contrairement à d’autres tissus, dans lesquels HIF-1α est. régulé à la hausse par activation transcriptionnelle via NF- kB, nous concluons que HIF-1α dans le testicule n’est. pas régulé à la hausse par une augmentation de Hif-1 αmRNA ou par des mécanismes NF-kB-dépendants. Les gènes de la voie hypoxie et les gènes impliqués dans le récepteur Toll-like (TLR) et dans la signalisation médiée par les cytokines comprennent des catégories fonctionnelles majeures de gènes régulés à la hausse, ce qui démontre qu’à la fois les voies de l’hypoxie et les voies classiques de l’inflammation sont impliquées dans les réponses inflammatoires du testicule. Mots-clés: Hypoxie, Orchite, Pathogènes, Lypopolysaccharides, Inflammation

Background of Leydig, Sertoli and spermatogenic cells [8, 9]. It is Infection and resulting inflammation of male reproduct- well-known that across species a number of antimicro- ive organs impairs fertility by decreasing sperm mobility bial peptides are produced by the testis including through the tract and reducing androgen production Spag11, members of the defensin family, Eppin and among other effects on spermatozoa and on the testis others [8, 10–17]. and epididymis [1–3]. Roughly half of all infertility cases Effects of uropathogenic bacteria and LPS in the testis are caused by male infertility and over 30% of male in- include inhibition of steroidogenesis and reduced andro- fertility is idiopathic, yet the relevance of infection and gen production [18, 19], decreased intracellular cAMP inflammation in male infertility, although widely studied and reduced sperm motility [20], induction of proinflam- clinically, is still subject to debate [4, 5]. Research to bet- matory cytokines and activation of antimicrobial path- ter understand the effects of pathogens on the male re- ways [10], and epigenetic regulation of antimicrobial productive tract and cellular and molecular mechanisms gene expression [21]. However, relatively little is known involved in the detection and response to infection of about interactions between regulatory pathways and male reproductive organs is essential. mechanisms involved in the response to inflammation of In vivo and in vitro models involving the administra- the testis. tion of lipopolysaccharide (LPS), a major pathogenic In other tissues, cross-talk between hypoxia regulatory component of the cell wall of Gram negative bacteria, is pathways and classic inflammatory pathways has been a common experimental approach to evaluate inflamma- demonstrated [22, 23]. This led us to hypothesize that tory responses [6, 7]. Pathogen-specific recognition sen- both pathways contribute significantly to inflammatory sors, such as Toll-like receptors (TLR), have been responses of the testis. We were intrigued by this hy- identified in the testis and implicated in the physiology pothesis in part because of our prior work on testis Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 3 of 12

hypoxia and the transcription factor hypoxia-inducible tissues were excised then frozen in liquid nitrogen and factor-1 (HIF-1). Recognized as the master regulator of stored at − 80 °C. hypoxia, HIF-1 is known to activate transcription of over 100 genes crucial for cellular responses to hypoxia [24– Serum testosterone assays 26]. Increasingly, HIF-1 activation has been implicated in Blood was collected via cardiac puncture at the time of a range of inflammatory responses [27]. For example, sacrifice, clotted at room temperature for 30 min, centri- HIF-1 is induced by proinflammatory cytokines such as fuged at 13,000 rpm for 10 min at 4 °C then sera was re- alpha (TNF-α) and interleukin beta moved and stored at − 80 °C prior to testosterone (IL-1ß) [28], and by LPS under normoxic conditions [29]. measurements. Testosterone radioimmunoassays were HIF-1α is expressed by Leydig cells in the normoxic adult carried out using a Packard 1900TR Liquid Scintillation rat testis and is unregulated by hypoxia [30]. We have also Analyzer (Canberra, Meriden, CT). Internal controls in- demonstrated that myeloid leukemia cell differentiation 1 cluded a no testosterone control and a positive control (Mcl-1) is a target gene for testicular HIF-1 with potentially sample of 100 ng/ml testosterone. important roles in antiapoptotic protection of Leydig cells [31]. Understanding mechanisms of HIF-1 activation under Protein extraction normoxic, hypoxic, and other physiological conditions, Frozen tissue samples were homogenized on ice in three such as inflammation, in the testis is of significant interest volumes of lysis buffer (10 mM Tris-HCL [pH 7.5], to investigators interested in the roles of HIF-1 on Leydig 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM Na3VO4 cell physiology. containing protease inhibitor cocktail; P8340; Sigma-Al- The present study was initiated to (1) investigate drich). The homogenate was maintained on ice for whether HIF-1 is affected by LPS-induced inflammation 10 min then centrifuged for 5 min at 3500 rpm at 4 °C. of the testis as an initial step to determine if co-activa- Supernatant was removed for cytoplasmic . Nu- tion of hypoxia and inflammatory pathways occurs in clei were resuspended in three volumes of 0.42 M KCl, the testis, (2) determine if other hypoxia pathway genes 20 mM Tris-HCL (pH 7.5), 1.5 mM MgCl2, 20% gly- are activated by inflammation, and (3) to explore poten- cerol, mixed for 30 min at 150 rpm at 4 °C, and centri- tial cross-talk between expression of hypoxia and inflam- fuged at 13,500 rpm for 30 min at 4 °C to isolate nuclear matory pathway genes. proteins. Protein extracts were stored at − 80 °C and protein concentrations determined by the Bradford assay (Bio-Rad, Hercules, CA). Methods Animals Immunoblot analysis Adult male, retired-breeder Sprague-Dawley rats (475– Proteins were separated by denaturing sodium dodecyl 750 g, 9–12 months, Charles River Laboratories (Stone sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Ridge, NY) were housed individually on a 12:12-h light/ through 7.5% PAGEr® Gold Precast Gels (Lonza, Rock- dark cycle and controlled temperature with free access land, MD) in 1× Tris-glycine SDS buffer (0.25 mM Tris, to food and water. 192 mM glycine, and 0.1% (w/v) SDS [pH 8.3]). COS-7 simian virus 40-transformed kidney cell nuclear extract Administration of Lipopolysaccharides (Active Motif, Carlsbad, CA) was included as a positive P. aeruginosa LPS (Type 10, L7018; Sigma-Aldrich, St. control for detecting HIF-1α. RAW 264.7 cell extract Louis, Missouri) and E. coli 055:B5 LPS (L2880; (Santa Cruz Biotechnology, Santa Cruz, CA) was in- Sigma-Aldrich) were selected because both are from cluded as positive control for NF-κB and IκBα. Proteins known pathogens of the urogenital tract and cause were electroblotted onto Trans-Blot nitrocellulose tissue-specific inflammatory responses. A dosage of (Bio-Rad), blocked with 1× Western wash (50 mM Tris, 5.0 mg/kg body weight was chosen to maximize activa- 30 mM NaCl, 0.001% Tween 20 [pH 7.6]) containing 5% tion of inflammatory responses. Others have clearly nonfat dry milk (NFDM) for 30 min at room demonstrated LPS doses that generate inflammatory re- temperature, then incubated in primary antibody over- sponses in vivo (1–5 mg/kg body weight) and in vitro night at 4 °C in 1× Western wash, 5% NFDM. (0.1–1.0 mg/ml), models [10, 16, 18, 19, 32]. LPS was Primary antibody dilutions were as follows: 0.5 μg/mL reconstituted in sterile 1× phosphate buffered saline HIF-1α mouse monoclonal antibody (AF1935; R&D Sys- (PBS; 10 mM sodium phosphate, 150 mM sodium chlor- tems, Minneapolis, MN), 0.5 μg/mL NF-κBrabbitpoly- ide, pH 7.8) and administered via intraperitoneal injec- clonal antibody (ADI-KAP-TF112, Enzo Life Sciences, tion. Sham controls were injected with sterile PBS. After Farmingdale, NY), 0.5 μg/mL IκB rabbit polyclonal anti- 1, 3, 6, and 12 h of treatment (n =5–7/time point), ani- body (9242; Cell Signaling, Danvers, MA). Actin was de- mals were euthanized via CO2 gas. Testes and control tected as a loading control for protein quantification using Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 4 of 12

a 1:2000 dilution of rabbit polyclonal antibody (A2066, Sigma-Aldrich). Blots were incubated in 1× Western wash, 5% NFDM containing appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:20,000 dilu- tion), washed in 1× Western wash, developed by enhanced chemiluminescence using either SuperSignal® West Pico substrate or SuperSignal® West Femto substrate (Pierce, Rockford, IL), and analyzed with a ChemiDoc™ XRS+ Mo- lecular Imager with Image Lab™ Software (Bio-Rad).

Gene expression profile analysis by real-time quantitative PCR (qPCR) Total RNA was isolated from frozen tissue using TRIzol reagent (Invitrogen, Grand Island, NY). Gene expression profiles for hypoxia pathway genes and inflammatory pathway genes were analyzed using Rat Hypoxia Signaling Fig. 1 Serum testosterone levels decrease following LPS-induced inflammation. *Indicates statistically significant difference as Pathway and Rat Innate and Adaptive Immune Response – 2 compared to sham (ANOVA, P <0.05;n =3 5); **Indicates P <0.001. Pathway RT Profiler™ PCR Arrays (Qiagen, Frederick, P.a., Pseudomonas aeruginosa MD). One microgram of genomic-DNA-free total RNA was reverse transcribed using the RT2 First Strand cDNA Kit (330,401, Qiagen). Real-time qPCR assays were carried protein expression data from other researchers. Infor- out with SYBR Green dye and amplified for 40 cycles in a mation about each gene of interest was gathered to Stratagene Mx3500P® thermocycler. Each array contained propose pathway maps. The Database for Annotation, 84 genes involved in the hypoxia or innate and adaptive Visualization, and Integrated Discovery (DAVID) Bio- immune response pathways. Listings of all genes queried informatics Resources 6.7 available through the Na- and accession numbers are available from the Qiagen tional Institute of Allergy and Infectious Diseases website. (NIAID) was utilized for functional annotation and Positive controls and housekeeping genes were in- pathway map analysis (https://david.ncifcrf.gov). cluded to normalize for differences in sample loading, genomic DNA contamination, and amplification effi- ciency. PCR controls included no RT and no template Electrophoretic mobility shift assays negative controls. Relative quantification of all target Electrophoretic Mobility Shift Assays (EMSA) were per- genes was calculated by first normalizing comparative formed using the Thermo Scientific LightShift® Chemilu- cycle threshold (Ct) values of target genes to ß-actin. minescent EMSA kit according to manufacturer’s Normalized values were used to calculate target gene ex- instructions (Thermo Scientific, Rockford, IL). Terminal pression in treatment groups compared to shams. Ct deoxynucleotidyl transferase (TdT) was used to catalyze values of controls were ~ 8–10 cycles beyond RT test biotin labeling of oligonucleotides (Biotin 3′ End DNA La- samples indicating that no contaminating DNA was beling Kit, Pierce). Binding reactions were carried out in present. Genes that displayed an average (n =4–7) mini- 20 μl volumes with protein extracts incubated with 10× mum of three-fold change (up- or down-regulation) binding buffer, 1 μg/μlpoly(dI•dC), 50% glycerol, 1% were considered statistically significant (p < 0.05) by ana- NP-40, 1 M KCl, 100 mM MgCl2, and 200 mM EDTA lysis of variance (ANOVA). and biotin-labeled oligonucleotide for 20 min at room temperature. Epstein-Barr Nuclear Antigen (EBNA) and In silico analysis biotin-labeled double-stranded target DNA were included Computer-based (in silico) methods were utilized for the as positive controls. Unlabeled EBNA oligonucleotides analysis of candidate genes involved in inflammatory re- were used in negative control and competition experi- sponses of the testis. Methods included literature ments. NF-κB double-stranded oligonucleotide with a searches using PubMed (http://www.ncbi.nlm.nih.gov/ consensus binding site for NF-κB /c-Rel homodimeric and pubmed/) and bioinformatics databases such as UniProt heterodimeric complexes (5’-AGTTGAGGGGACTTTCC (https://www.uniprot.org) for gene expression and pro- CAGGC -3′; sc-2505, Santa Cruz Biotechnology) which is tein distribution data. These electronic resources were similar to the sequence present in the upstream region of used to determine if there was existing data available the HIF-1 gene and mutant oligonucleotides (sc-2511) about these genes, such as expression and regulation with a C-G substitution in the consensus site were used data, cell-type-specific expression in the testis, and for EMSA. Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 5 of 12

AB

Fig. 2 HIF-1α protein levels increase following LPS-induced inflammation in rat testis. a Results of HIF-1α immunoblot analysis of testicular cytoplasmic proteins isolated from sham and LPS treated animals. COS-7 cell line protein extract was included as a positive control and ß-actin was included as an internal control. HIF-1α was detected at ~ 120 kDa. Shown is a 6 h sham as an example but time-matched shams (1, 3 and 6 h) were used for all experiments and for quantitation. Representative time points and results are shown in panel (a). b Histogram shows relative HIF-1α protein levels normalized to ß-actin and as compared to sham for all time points and treatment samples analyzed. *Indicates statistically significant difference as compared to sham (ANOVA, P < 0.05; n =4–7). **Indicates P < 0.001

EMSA reactions were separated through 6% polyacryl- physiological effect of LPS inflammation on the testis amide TBE PAGEr® Gold gels (Lonza) electrophoresed in under our experimental conditions. 0.5× tris-borate-EDTA buffer (AccuGENE® 10× TBE Buffer, Increased expression of Il-6 has been reported in the Lonza), transferred onto nylon (Biodyne® B, Thermo Scien- testis following LPS-induced inflammation at both 3 and tific) and membranes cross-linked at 120 mJ/cm2 for 60 s 9-h time points [21]. As mentioned later in the manu- using a UV-light cross-linker. Blots were blocked, incubated script, we detected elevated mRNA expression for Il-6 with a 1:300 dilution of stabilized streptavidin-HRP conju- mRNA in 3 h LPS treatment groups providing further gate for 15 min at room temperature, then reacted with lu- evidence for inflammation of the testis under these ex- minal/enhancer solution. Digital images were captured with perimental conditions (see Table 2). aChemiDoc™ XRS+ quantitative analysis performed using Image Lab™ Software. HIF-1α protein levels increase following LPS-induced inflammation Results To determine if testis inflammation results in the activa- Serum testosterone levels decrease following LPS tion of hypoxia pathways through HIF-1, HIF-1α protein treatment levels in testis from sham and treatment groups were de- A rat model of orchitis, or testicular inflammation, was tected by Western blot analysis (Fig. 2a). Statistically sig- established via administration of P. aeruginosa or E. coli nificant increases in HIF-1α protein were observed at 1 h, LPS at a dosage of 5 mg/kg bw. It is well-established that 3hand6hP. aeruginosa LPS treatment as compared to one effect of LPS on the testis is diminished testosterone sham and following 6 h E. coli LPS treatment (Fig. 2b). In output and reductions in plasma testosterone levels [19]. an initial pilot study to determine time course and LPS To determine if 1 h, 3 h, and 6 h P. aeruginosa and 6 h dosage, 1 h and 3 h E. coli LPS treatment experiment was E. coli LPS treatment caused acute inflammation of the performed but no apparent changes in HIF-1α protein testis and the expected reduction in circulating testoster- were detected (data not shown), therefore it appears that one, serum testosterone levels were measured via radio- P. aeruginosa LPS produces a more acute phase response immunoassay. No decrease in serum testosterone levels in elevating HIF-1 than E. coli LPS. was observed at 1 h P. aeruginosa LPS treatment (Fig. 1). Statistically significant decreases in serum testosterone NF-κB and IκB protein levels following LPS-induced levels were observed at 3 h and 6 h P. aeruginosa and inflammation 6hE. coli LPS treatment as compared to sham (P < 0.05; NF-κB is a major activator of inflammatory pathways and Fig. 1), demonstrating the expected and well-reported has been shown to transcriptionally regulate HIF-1 under Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 6 of 12

A

B C

Fig. 3 Phospho-NF-κB and IκB protein levels do not change following LPS-induced inflammation in rat testis. a Results of phospho-NF-κB and phospho-IκB immunoblot analysis of testicular cytoplasmic proteins isolated from sham and LPS treated animals. RAW cell line protein extract was included as a positive control and ß-actin was included as an internal control. Representative time points and results are shown in panel (a). Histograms show relative phospho-IκB(b) and phospho-NF-κB(c) protein levels normalized to ß-actin and as compared to sham for all time points and treatment samples analyzed. No statistically significant differences in phospho-IκB and phospho-NF-κB protein levels were observed between sham and LPS treated groups (ANOVA, P < 0.05; n =3–5). STD, protein molecular weight standards conditions of inflammation in other cell types [33, 34]. In different treatments. A decrease in binding activity was an effort to identify the mechanism(s) responsible for observed in the cytoplasmic extracts of both 3 h P. aeru- HIF-1α protein accumulation following LPS-induced in- ginosa and 6 h E. coli LPS treatment and nuclear ex- flammation of the testis, phospho-NF-κB and phospho-IκB tracts of 6 h E. coli LPS treatment as compared to sham protein levels in testicular cytoplasmic protein extracts were (P<0.05; Fig. 4b). measured via Western blot analysis. No differences in phospho-NF-κB and phospho-IκB protein levels were ob- Gene expression profiling of hypoxia pathway genes served in the testis following LPS-induced inflamma- That HIF-1 increased following LPS-induced inflammation tion at all time points as compared to sham (P < 0.05; suggests roles for hypoxia regulated genes in inflammatory Figs. 3a-c). responses of the testis. Gene expression profiling arrays were used to identify hypoxia pathway genes that are regu- NF-κB DNA binding activity decreases following LPS- lated following LPS administration. Also, to determine if induced inflammation regulation of HIF-1α protein accumulation following EMSAs were performed to evaluate the DNA binding LPS-induced inflammation of the testis occurs at the tran- activity of NF-κB. An oligonucleotide with a consensus scriptional level, Hif-1α mRNA levels were measured in binding site for NF-κB homo- and heterodimeric com- these arrays. No statistical significant difference in Hif-1α plexes, similar in sequence to a binding site upstream of mRNA levels was observed at any time point following the HIF-1 gene, was used for EMSA with both testicular LPS-induced inflammation in the testis by the arrays or by cytoplasmic and nuclear protein extracts (Fig. 4a). These independent RT-PCR assays for Hif-1α (data not shown), time points were chosen to determine if there was any suggesting that HIF-1 protein up-regulation is occurring at difference in inflammatory response caused by the the post-transcriptional level. Stabilization and degradation Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 7 of 12

AB

Fig. 4 Electrophoretic mobility shift assay (EMSA) for NF-κB binding to the HIF-1α gene. a NF-κB binding to a consensus sequence from the HIF-1α gene in both cytoplasmic and nuclear protein extracts decreased following LPS treatment. b Relative DNA binding activity as compared to sham. *Indicates statistically significant difference as compared to sham (ANOVA, n =3–5; P < 0.05). **Indicates P<0.001 of HIF-1 protein is very well characterized as the primary LPS-stimulated genes indicates that these transcripts are mechanism controlling HIF-1 protein levels [26]. predominantly expressed in supporting cells of the testis Pathway array results demonstrated that 5 genes in the (Sertoli, myoid, and Leydig cells) and not in developing hypoxia pathway, Angptl4, Egr1, Ier3, Pai1, and Slc2a1 germ cells. Egr1, Fos, Ier3, and Pai1 are known target (Glut1), were up-regulated after 3 h of LPS-induced in- genes for the transcription factor NF-κB. None of the flammation and expression and showed more than a up-regulated genes are known HIF-1 targets. three-fold change (p < 0.05; Table 1). Following 6 h of LPS-induced inflammation, 3 of these genes (Angptl4, Gene expression profiling of innate and adaptive immune Bhlhe40, and Egr1), showed statistically significant response pathway genes up-regulation (Table 1). No genes were down-regulated Because recent research has demonstrated cross-talk be- following LPS administration. In silico analysis of tween hypoxia pathways and inflammatory pathways, we

Table 1 Up-regulation of hypoxia pathway genes after 3 and 6 h of LPS-induced inflammation Gene Name Unigene, GenBank Fold Change 3 h (n = 7) 6 h (n = 6) Angiopoietin-like 4 (Angptl4)Rn.119611, NM_199115 20.38 (p = 0.007) 14.12 (p = 0.005) Basic helix-loop-helix family, member (Bhlhe40) No change 3.31 Rn.81055, NM_053328 (p = 0.000) Early growth response 1 (Egr1) 29.16 6.67 Rn.9096, NM_012551 (p = 0.006) (p = 0.046) Immediate early response 3 (Ier3) 9.85 No change Rn.23638, NM_212505 (p = 0.001) Plasminogen activator inhibitor type 1 (Pai1) 15.05 No change Rn.29367, NM_012620 (p = 0.023) Solute carrier family 2 (facilitated glucose transporter), 3.41 No change member 1 (Slc2a1, Glut1) (p = 0.002) Rn.3205, NM_138827 Only genes that showed at least a three-fold increase in expression with p < 0.05 were considered statistically significant Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 8 of 12

chose to also evaluate expression profiles for innate and Pathway analysis of inflammatory genes affected by adaptive immune response genes. At 3 h following P. LPS-induced inflammation aeruginosa LPS treatment, 11 genes showed a statisti- DAVID Bioinformatics Resources 6.7 was utilized for cally significant up-regulation (Cc112, Cc13, Cd14, functional annotation and pathway map analysis. This Cxc110, Icam1, Il10, Il1b, Il6, Nfkbia, Tlr2, and Tnf) and allowed for identification of multiple genes involved in one gene was down-regulated (Tlr5; Table 2). Twelve specific signaling pathways. Of the inflammatory genes genes were up-regulated at 6 h (C3, Cc112, Cc13, Cd14, affected by LPS, 9 genes are part of the TLR signaling Il1b, Jak2, Nfkbia, Nlrp3, Slc11a1, Tlr1, Tlr2, and Tnf). pathway (Additional file 1: Figure S1). With the excep- Seven genes remained elevated at both 3 and 6 h tion of Tlr5, which was down-regulated 3 h following (Cc112, Cc13, Cd14, Il1b, Nfkbia, Tlr2, and Tnf). LPS-induced inflammation, genes circled in red were

Table 2 Changes in expression of genes involved in innate and adaptive immune responses after 3 and 6 h of LPS-induced inflammation Gene Name Fold Change Gene Functions 3h 6h Complement component 3 (C3) No change 7.72 Activation of the complement system. Rn.11378, NM_016994 (p = 0.002) Chemokine (C-C motif) ligand 12 8.52 26.15 Angiogenesis; cellular response to fibroblast growth (Cc112); Rn.137780, NM_001105822 (p = 0.000) (p = 0.035) factor stimulus; participates in chemokine and cytokine mediated and cytokine signaling pathways. Chemokine (C-C motif) ligand 3 (Cc13) 10.62 14.63 Monokine with inflammatory and chemokinetic properties. Rn.10139, NM_013025 (p = 0.000) (p = 0.043) CD 14 molecule (Cd14) 7.17 6.67 Accessory protein for TLR4. Rn.42942, NM_021744 (p = 0.002) (p = 0.002) Chemokine (C-X-C motif) ligand 10 24.23 NSS Proinflammatory cytokine; may participate in T-cell effector (Cxc110); Rn.10584, NM_139089 (p = 0.000) function and development. Inhibits expression of StAR D1. Intercellular adhesion molecule 1 25.26 NSS Up-regulated by TLR2, TLR4, TLR5, and TLR6; ligand for (1cam1) Rn. 12, NM_012967 (p = 0.000) leukocyte adhesion protein. Interleukin 10 (1110) 4.43 No change Inhibits cytokine synthesis. Rn.9868, NM_012854 (p = 0.021) Interleukin 1 beta (111b) 11.32 14.18 Endogenous pyrogen, proinflammatory cytokine. Rn.9869, NM_031512 (p = 0.000) (p = 0.053) Interleukin 6 (116) 69.61 NSS Disrupts integrity of blood-testis-barrier by altering steady Rn.9873, NM_012589 (p = 0.000) state levels of membrane proteins. Janus kinase 2 (Jak2) No change 3.77 Non-receptor tyrosine kinase known to be involved in innate Rn.18909, NM_031514 (p = 0.032) and adaptive immunity signaling. Nuclear factor of kappa light polypeptide 4.84 3.99 Inhibitor of NF-ĸB; binds NF-ĸB and retains it in the cytoplasm. gene (Nfkbia) (p = 0.000) (p = 0.018) Rn.12550, NM_001105720 NLF family, pyrin domain containing No change 8.27 No functional assignments in Uniprot. 3 (Nlrp3) (p = 0.001) Rn.214177, XM_220513 Solute carrier family 11 (proton-coupled No change 3.79 Macrophage protein associated with resistance or susceptibility divalent; Slc11a1) (p = 0.000) to intracellular pathogens. Rn.105919, NM_001031658 Toll-like receptor 1 (Tlr1) No change 3.36 Stimulated by bacterial lipoproteins. Rn. 107,212, NM_001172120 (p = 0.018) Toll-like receptor 2 (Tlr2) 6.35 8.70 Increase expression of inflammatory cytokines IL-α, IL-6, and Rn. 46,387, NM_198769 (p = 0.000) (p = 0.027) interferon-α, and β Toll-like receptor 5 (Tlr5) −3.37 No change Increase expression of inflammatory cytokines IL-α, IL-6, and Rn. 198,962, NM_001145828 (p = 0.052) interferon-α, and β Tumor necrosis factor (TNF superfamily, 8.78 4.58 Chemokine-mediated signaling, induces germ cell in member 2; Tnf) (p = 0.000) (p = 0.029) autoimmune orchitis. Rn.2275,NM_012675 Genes were reported that showed more than a three-fold change following LPS-induced inflammation with p < 0.05. NSS, not statistically significant, greater than three-fold change observed with p > 0.05 Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 9 of 12

up-regulated at 3 and/or 6 h following LPS administra- been demonstrated [36] and LPS induces HIF-1 activa- tion (Additional file 1: Figure S1). tion in human monocytes via NF-κB[33]. This led us to consider that NF-κB may be an important transcriptional activator of HIF-1α following LPS-induced Discussion inflammation in our model. EMSA experiments to meas- This study was designed to begin to understand effects ure NF-κB binding to the HIF-1α promoter revealed that of E. coli and P. aeruginosa LPS-induced inflammation binding activity decreased following LPS-induced inflam- in activating HIF-1 and hypoxia pathway gene responses mation. Nfkb1 mRNA and protein expression did not in- in the testis. Regulation of HIF-1 during inflammation crease and our pathway array results also demonstrated has been studied in vivo and in vitro and reviewed ex- increased expression of Nfkbia mRNA. Taken together tensively [23]. One hypothesis is that HIF-1 is these results suggest that NF-κBisprobablynotatran- up-regulated in microenvironments of inflamed tissues scriptional activator of HIF-1α in the testis in vivo. characterized by low levels of oxygen and glucose and We also demonstrated that testicular HIF-1α is not high levels of inflammatory cytokines, reactive oxygen up-regulated through an increase in Hif-1α mRNA. It is species (ROS), and nitrogen species, to protect cells likely then that post-translational regulation of HIF-1α against secondary inflammatory damage [32]. protein levels (via ubiquitination and protein stability) as Steroidogenesis and spermatogenesis are inhibited by occurs under hypoxic conditions, and not transcriptional infection and inflammation of the testis [18]. For ex- regulation of HIF-1α is the likely mechanism for ample, in mice using relatively high doses of LPS, Leydig up-regulating HIF-1α protein levels in the testis during cell steroidogenesis is inhibited via reduced synthesis of inflammation. the cholesterol transport protein steroidogenic acute One of the functional clusters of inflammatory genes regulatory protein and steroidogenic enzymes [19]. To up-regulated in our study is the TLRs. In our work ex- validate inflammation in our model system, serum tes- pression of Tlr2 was shown to be elevated at both 3 and tosterone levels were measured and decreases in serum 6-h time points but Tlr4 mRNA was not. Although testosterone after the 3 h and 6 h P. aeruginosa and 6 h others have demonstrated elevated expression of TLR-4 E. coli LPS treatments confirmed LPS-induced inflam- following LPS challenge of the testis [10]. But the ab- mation and compromised testicular functions. sence of significant change in expression of Tlr4 has Previously we have shown that HIF-1 is highly been reported before. For example, Winnall et al. re- expressed by normoxic Leydig cells and that, unlike ported a significantly higher expression of TLR2 in rela- other tissues, testicular HIF-1 expression is not induced tion to expression of the LPS receptor TLR4 and its by hypoxia [35]. This may be because physiologic oxygen co-receptor protein CD14 [7]. Sertoli cells have been tension of the testis is already hypoxic. Thus, studying shown to respond to bacteria through both TLR2 and the activation of a hypoxic response during inflamma- TLR4 [39]. TLR4-dependent activation of HIF-1 in the tion in what may be an already hypoxic environment is lung has been demonstrated [40]. Our studies showed ele- of particular interest. In vitro studies have established vated expression of CD14. In addition, TLR stimulation el- the activation of hypoxia genes by LPS in multiple sys- evates the synthesis of proinflammatory cytokines such as tems including pulmonary artery smooth muscle cells, IL-1β and TNF (both of which showed elevated mRNA macrophages, monocytes, and glioblastomas [33, 34, 36– expression in our study) so perhaps TLR-dependent path- 38]. The increase in testicular HIF-1 protein we detected ways and not NF-κB is the predominant activation path- following LPS-induced inflammation indicates a role for way for HIF-1 in the testis. Recently we have also HIF-1 and Leydig cells in the activation of (or protection demonstrated that expression of a number of microRNAs against) an inflammatory response. in the testis are downregulated by LPS including miRNAs The accumulation of testicular HIF-1α protein follow- involved in regulating the NF-κBpathway[41]. ing inflammation suggests crosstalk between inflamma- Testicular macrophages are important for modulating tory responses and hypoxic responses in the testis. inflammatory responses [1]. In testicular macrophages Before determining the effects of HIF-1 up-regulation challenged with LPS, it has been shown that the NF-κB following LPS-induced inflammation, we chose to make signaling pathway is blocked due to a lack of IκBα ubi- an initial effort to identify potential pathways responsible quitination and degradation [42]. Rather LPS stimulates for the crosstalk between inflammatory and hypoxic re- testicular macrophages to activate MAPK, AP-1, CREB sponses and up-regulation of HIF-1α. Studies suggest and other pathways that increase production of proin- this crosstalk occurs via transcriptional regulation of flammatory cytokines such as IL-6 and TNF. Both IL-6 HIF-1α by NF-κB, a key transcription factor responsible and TNF are known to have inhibitory roles on steroido- for mediating inflammatory responses [34, 37]. A func- genesis of Leydig cells [1, 43] and so the elevated mRNA tional NF-κB binding site in the HIF-1α promoter has expression of these genes observed in our studies was Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 10 of 12

expected. In this study up-regulated expression of known target genes of NF-κβ. Also, although HIF-1 is mRNAs for proinflammatory cytokines such as IL-1 and known to activate over 100 target genes, not all tar- IL-6, TNF underscores the importance of cytokine-medi- gets were built into the array; therefore, we cannot ated signaling pathways that are thought to be important exclude the possibility that there may be other HIF-1 for inflammatory responses of the testis, as target genes involved in inflammation. demonstrated by other investigators [1]. Finally, the up-regulation of HIF-1α protein may also Conclusions be caused by several physiologic changes. For example, a Regardless of the underlying reasons for HIF-1 activa- decrease in oxygen tension caused by inflammatory ac- tion, we conclude that the mechanism of HIF-1α protein tivity occurring in the testis may result in a hypoxic en- accumulation following LPS-induced inflammation is vironment leading to HIF-1 activation. Similarly ROS not at the transcriptional level and likely not mediated released during inflammatory responses can also through NF-κB. Further studies will be carried out to stabilize and elevate HIF-1α protein levels [23]. identify target genes regulated by HIF-1. This study pro- Results from hypoxia pathway arrays demonstrated vides a baseline analysis of hypoxia and inflammatory that six hypoxic pathway genes (Angptl4, Bhlhe40, Egr1, pathway gene expression changes following LPS-induced Ier3, Pai1, and Glut1) are up-regulated 3 or 6 h follow- inflammation of the testis that will be a useful resource ing LPS-induced inflammation. Among 84 hypoxic for researchers in the future. The range of genes affected genes, no genes appear to be down-regulated in re- and their potential functions, reflects the complexity of sponse to LPS. Those that did show a significant in- the inflammatory response of the testis and underscores crease do not appear to belong to any particular that both hypoxic and inflammatory gene expression functional cluster. Due to the variety of functions exhib- pathways are involved. ited by these genes, we do not want to speculate about the roles these genes may plan in inflammatory re- sponses in the testis. These genes are the subject of fu- Additional file ture studies. Although we did not determine the cell-type specific Additional file 1: Figure S1. Toll-like receptor signaling pathway including innate and adaptive immune response genes up-regulated or down- location of the genes analyzed in our study, in silico ana- regulated by LPS-induced inflammation. (DOCX 359 kb) lysis of hypoxia pathways genes revealed that only Bhlhe40 is known to be expressed in spermatogenic Abbreviations cells. Angptl4, Egr1, Ier3, and Pai1 are expressed in ANOVA: analysis of variance; cAMP: cyclic AMP; cDNA: complementary DNA; myoid cells. Pai1 is expressed in germ cells. Egr1, Ier3, Ct: cycle threshold; DAVID: Database for Annotation, Visualization, and Pai1, and Slc2a1 are expressed in Sertoli cells, and Egr1 Integrated Discovery; DNA: deoxyribonucleic acid; EBNA: Epstein-Barr Nuclear Antigen; EBNA: Epstein-Barr Nuclear Antigen; EDTA: ethylene-Diamine-Tetra- is expressed in Leydig cells. These results suggest that Acetic acid; EMSA: Electrophoretic Mobility Shift Assays; HIF-1α: hypoxia- supporting cells, and not germ cells, are primarily in- inducible factor-1 alpha; HRP: horseradish peroxidase; IL-1ß: interleukin beta; κ α volved in inflammatory responses of the testis. I B : inhibitory NFKB alpha; LPS: lipopolysaccharide; Mcl-1: myeloid leukemia cell differentiation 1; NFDM: nonfat dry milk; NIAID: National Institute of In addition, the literature includes few studies on hyp- Allergy and Infectious Diseases; PBS: phosphate buffered saline; oxic gene expression in the testis in response to LPS. PCR: polymerase chain reaction; qPCR: real-Time Quantitative PCR; Bourdon et al. detected Pai1 expression in cultured Ser- RNA: ribonucleic acid; ROS: reactive oxygen species; RT: reverse transcriptase; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; toli cells following LPS treatment [44]. Further experi- TdT: terminal deoxynucleotidyl transferase; TLR: Toll-like receptor; TNF- mental evidence is necessary to elucidate the specific α: tumor necrosis factor alpha mechanisms and functions of these genes, and will pro- vide a greater understanding of why some appear to be Acknowledgements We thank Dr. Renshan Ge and Chantal Sottas at the Population Council of early response genes while others may be late re- the Center for Biomedical Research for performing serum testosterone sponders. These studies could also provide an explan- radioimmunoassays. Results from this study were presented at the 34th ation for why certain genes such as Angptl4 and Egr1 annual meeting of the American Society for Reproductive Immunology (Long Beach, NY, June 2014), the 39th annual meeting of the American show a sustained response, with elevated expression at Society of Andrology (Atlanta, GA, April 2014), the 112th Annual Meeting of both 3 and 6-h time points. the American Society for Microbiology (San Francisco, CA, June 2012) and While the up-regulation of HIF-1α suggests a role the 37th Annual Meeting of the American Society of Andrology (Tucson, AZ, April 2012). for the HIF-1 pathway in inflammatory responses of the testis, further studies are needed to determine Funding possible HIF-1 target genes. HIF-1 was initially Supported by NIH grant R15-HD046451 to M. Palladino, the Independent thought to play a significant role in the regulation of College Fund of New Jersey-Merck Undergraduate Science Endeavors Program to support G. Fasano, and the Bristol-Myers Squibb Charitable these hypoxic genes, but only Glut1 is a known target Giving Grant to the Monmouth University Summer Research Program to gene for HIF-1. However, Egr1, Ier3, and Pai1 are support D. Patel. Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 11 of 12

Availability of data and materials 14. Wang Z, Widgren EE, Sivashanmugam P, O’Rand MG, Richardson RT. All data generated or analyzed during this study are included in this Association of eppin with semenogelin on human spermatozoa. Biol published article [and its Additional files]. Reprod. 2005;72:1064–70. 15. Yenugu S, Chintalgattu V, Wingard CJ, Radhakrishnan Y, French FS, Hall SH. Authors’ contributions Identification, cloning and functional characterization of novel beta- MAP designed the study, participated in its experimental design, data analysis, defensins in the rat (Rattus norvegicus). Reprod Biol Endocrinol. 2006;4:7. and drafting and final writing and submission of the manuscript. GAF carried 16. Rodrigues A, Queiróz DB, Honda L, Silva EJ, Hall SH, Avellar MC. Activation out hypoxia and inflammatory pathway array experiments and drafting of the of toll-like receptor 4 (TLR4) by in vivo and in vitro exposure of rat manuscript. DP carried out western blot experiments and drafting of the epididymis to lipopolysaccharide from Escherichia Coli. Biol Reprod. 2008;79: manuscript. CD and ML carried out EMSA experiments and drafting of the 1135–47. manuscript. All authors read and approved the final manuscript. 17. Hall SH, Hamil KG, French FS. Host defense proteins of the male reproductive tract. J Androl. 2002;23:585–97. Ethics approval and consent to participate 18. O’Bryan MK, Schlatt S, Phillips DJ, De Kretser DM, Hedger MP. Bacterial Animal research protocols were approved by the Monmouth University lipopolysaccharide-induced inflammation compromises testicular function IACUC and conform to guidelines established in The Guide for the Care at multiple levels in vivo. Endocrinology. 2000;141:238–46. and Use of Laboratory Animals. 19. Allen J, Diemer T, Janus P, Hales K, Hales B. Bacterial endotoxin lipopolysaccharide and reactive oxygen species inhibit leydig cell Consent for publication steroidogenesis via perturbation of mitochondria. Endocrine. 2004;25:265–75. Not applicable. 20. Li Z, Zhang D, He Y, Ding Z, Mao F, Luo T, et al. Lipopolysaccharide compromises human sperm function by reducing intracellular cAMP. Competing interests Tohoku J Exp Med. 2016;238:105–12. The authors declare that they have no competing interests. 21. Biswas B, Yenugu S. Lipopolysaccharide induces epididymal and testicular antimicrobial gene expression in vitro: insights into the epigenetic Publisher’sNote regulation of sperm-associated antigen 11e gene. Immunogenetics. 2013;65: 239–53. Springer Nature remains neutral with regard to jurisdictional claims in published 22. Bartels K, Grenz A, Eltzschig HK. Hypoxia and inflammation are two sides of maps and institutional affiliations. the same coin. Proc Natl Acad Sci U S A. 2013;110:18351–2. 23. Devraj G, Beerlage C, Brüne B, VAJ K. Hypoxia and HIF-1 activation in Received: 30 July 2018 Accepted: 15 October 2018 bacterial infections. Microbes Infect. 2017;19:144-56. 24. Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell. – References 2012;148:399 408. 1. Hedger MP. The Immunophysiology of male reproduction. Knobil and 25. Yee Koh M, Spivak-Kroizman TR, Powis G. HIF-1 regulation: not so easy – Neill’s physiology of reproduction. 4th ed. London: Academic Press; 2015. come, easy go. Trends Biochem Sci. 2008;33:526 34. 2. Azenabor A, Ekun AO, Akinloye O. Impact of inflammation on male 26. Brocato J, Chervona Y, Costa M. Molecular responses to hypoxia-inducible α – reproductive tract. J Reprod Infertil. 2015;16:123–9. factor 1 and beyond. Mol Pharmacol. 2014;85:651 7. 3. Bachir BG, Jarvi K. Infectious, inflammatory, and immunologic conditions 27. Dehne N, Brüne B. HIF-1 in the inflammatory microenvironment. Exp Cell – resulting in male infertility. Urol Clin North Am. 2014;41:67–81. Res. 2009;315:1791 7. α 4. Ochsendorf FR. What are the consequences of sexually transmitted 28. Tewari R, Choudhury SR, Ghosh S, Mehta VS, Sen E. Involvement of TNF - κ α infections on male reproduction? Handbook of Andrology. Lawrence: Allen induced TLR4-NF- B and TLR4-HIF-1 feed-forward loops in the regulation – Press, Inc; 2009. of inflammatory responses in glioma. J Mol Med. 2012;90:67 80. 5. Hamada A, Esteves SC, Agrawal A. The role of contemporary andrology in 29. Imtiyaz HZ, Simon MC. Hypoxia-inducible factors as essential regulators of – unraveling the mystery of unexplained male infertility. Open Reprod Sci J. inflammation. Curr Top Microbiol Immunol. 2010;345:105 20. 2011;3:27–41. 30. Powell JD, Elshtein R, Forest DJ, Palladino MA. Stimulation of hypoxia- α 6. Hedger M, Klug J, Fröhlich S, Müller R, Meinhardt A. Regulatory cytokine inducible Factor-1 alpha (HIF-1 ) protein in the adult rat testis following expression and interstitial fluid formation in the normal and inflamed rat ischemic injury occurs without an increase in HIF-1α messenger RNA testis are under leydig cell control. J Androl. 2005;26:379–86. expression. Biol Reprod. 2002;67:995–1002. 7. Winnall WR, Muir JA, Hedger MP. Differential responses of epithelial Sertoli 31. Palladino MA, Shah A, Tyson R, Horvath J, Dugan C, Karpodinis M. Myeloid cells of the rat testis to toll-like receptor 2 and 4 ligands: implications for cell leukemia-1 (Mc1–1) is a candidate target gene of hypoxia-inducible studies of testicular inflammation using bacterial lipopolysaccharides. Innate factor-1 (HIF-1) in the testis. Reprod Biol Endocrinol. 2012;10:104. Immun. 2011;17:123–36. 32. Frede S, Berchner-Pfannschmidt U, Fandrey J. Regulation of hypoxia- 8. Palladino M, Johnson T, Gupta R, Chapman J, Ojha P. Members of the toll- inducible factors during inflammation. Meth Enzymol. 2007;435:405–19. like receptor family of innate immunity pattern-recognition receptors are 33. Frede S, Stockmann C, Freitag P, Fandrey J. Bacterial lipopolysaccharide abundant in the male rat reproductive tract. Biol Reprod. 2007;76:958–64. induces HIF-1 activation in human monocytes via p44/42 MAPK and NF- 9. Girling JE, Hedger MP. Toll-like receptors in the gonads and reproductive kappaB. Biochem J. 2006;396:517–27. tract: emerging roles in reproductive physiology and pathology. Immunol 34. Gorlach A, Bonello S. The cross-talk between NF-κB and HIF-1: further Cell Biol. 2007;85:481–9. evidence for a significant liaison. J Biochem. 2008;412:e17. 10. Biswas B, Yenugu S. Antimicrobial responses in the male reproductive 35. Palladino MA, Pirlamarla PR, McNamara J, Sottas CM, Korah N, Hardy MP, et tract of lipopolysaccharide challenged rats. Am J Reprod Immunol. al. Normoxic expression of hypoxia-inducible factor 1 in rat Leydig cells in 2011;65:557–68. vivo and in vitro. J Androl. 2011;32:307–23. 11. Palladino MA, Mallonga TA, Mishra MS. Messenger RNA (mRNA) expression 36. Bonello S, Zähringer C, BelAiba RS, Djordjevic T, Hess J, Michiels C, et al. for the antimicrobial peptides beta-defensin-1 and beta-defensin-2 in the Reactive oxygen species activate the HIF-1alpha promoter via a functional male rat reproductive tract: beta-defensin-1 mRNA in initial segment and NFkappaB site. Arterioscler Thromb Vasc Biol. 2007;27:755–61. caput epididymidis is regulated by androgens and not bacterial 37. Rius J, Guma M, Schachtrup C, Akassoglou K, Zinkernagel AS, Nizet V, et al. lipopolysaccharides. Biol Reprod. 2003;68:509–15. NF-|[kgr]|B links innate immunity to the hypoxic response through 12. Biswas B, Narmadha G, Choudhary M, French FS, Hall SH, Yenugu S. transcriptional regulation of HIF-1|[agr]|. Nature. 2008;453:807–11. Identification of toll-like receptors in the rat (Rattus norvegicus): messenger 38. Blouin CC, Pagé EL, Soucy GM, Richard DE. Hypoxic gene activation by RNA expression in the male reproductive tract under conditions of lipopolysaccharide in macrophages: implication of hypoxia-inducible factor androgen variation. Am J Reprod Immunol. 2009;62:243–52. 1α. Blood. 2004;103:1124–30. 13. Com E, Bourgeon F, Evrard B, Ganz T, Colleu D, Jégou B, et al. Expression of 39. Hedger MP. Toll-like receptors and signalling in spermatogenesis and antimicrobial defensins in the male reproductive tract of rats, mice, and testicular responses to inflammation—a perspective. J Reprod Immunol. humans. Biol Reprod. 2003;68:95–104. 2011;88:130–41. Palladino et al. Basic and Clinical Andrology (2018) 28:14 Page 12 of 12

40. Zhou Z, Zhu X, Chen J, Yang S, Sun R, Yang G. The interaction between toll- like receptor 4 signaling pathway and hypoxia-inducible factor 1α in lung ischemia-reperfusion injury. J Surg Res. 2014;188:290–7. 41. Parker MI, Palladino MA. MicroRNAs downregulated following immune activation of rat testis. Am J Repro Immunol. 2017;00:e12673. 42. Bhushan S, Tchatalbachev S, Lu Y, Fröhlich S, Fijak M, Vijayan V, et al. Differential activation of inflammatory pathways in testicular macrophages provides a rationale for their subdued inflammatory capacity. J Immunol. 2015;194:5455–64. 43. Hales DB. Testicular macrophage modulation of Leydig cell steroidogenesis. J Reprod Immunol. 2002;57:3–18. 44. Bourdon V, Defamie N, Fenichel P, Pointis G. Regulation of tissue-type plasminogen activator and its inhibitor (PAI-1) by lipopolysaccharide- induced phagocytosis in a Sertoli cell line. Exp Cell Res. 1999;247:367–72.